The probability of curing cancers, through surgery alone, is high in those individuals whose primary tumors are detected at a relatively early stage. Such early detection is therefore one of the most promising approaches for limiting cancer morbidity and mortality in the future (1). At present, PAP smears can be used to detect cervical cancers, mammography can detect breast cancers, serum PSA levels can signify the presence of prostate cancer, and colonoscopy and fecal occult blood tests can detect colon cancers (2). However, problems in sensitivity, specificity, cost or compliance have complicated widespread implementation of these tests (3-5). Moreover, methods for the early detection of most other cancer types are not yet available.
The discovery of the genetic bases of neoplasia has led to new approaches to detect tumors non-invasively (6-8). Many of these approaches rely on the ex vivo detection of mutant forms of the oncogenes and tumor suppressor genes that are responsible for the initiation and progression of tumors. This approach was first used to detect bladder and colon tumors through examination of urine and stool, respectively (9, 10), and has since been used to detect several other tumor types (11-14). As the mutant genes are not only “markers” for cancer, but are the proximate causes of tumor growth (1), they have major conceptual advantages over conventional markers such as fecal occult blood or serum PSA. In particular, conventional markers are not pathogenically involved in the tumorigenic process and are much less specific for neoplasia than are mutations.
The evaluation of patient blood samples for mutant DNA molecules is a particularly attractive approach as such tests could detect many different forms of cancers. Additionally, blood can be easily obtained from patients during routine outpatient visits and methods for preparing and storing plasma and serum are well-known and reliable. Accordingly, numerous studies have attempted to identify abnormal forms or quantities of DNA in plasma or serum (6, 11-15). Unfortunately, the results of many of these studies are contradictory. Some report high detection rates of cancers, others very low, despite the use of similar techniques and patient cohorts. Moreover, several studies have shown that loss of heterozygosity is routinely detectable in circulating DNA, even in patients with relatively non-aggressive tumors. To detect loss of heterozygosity in such samples, the neoplastic cells within a tumor must contribute more than 50% of the total circulating DNA.
The prior studies, though promising, lead to several questions that must be answered to engender confidence in the use of circulating, abnormal DNA as a biomarker of malignancy. First, how many copies of a given gene fragment are present in the circulation in cancer patients? Second, what is the nature of this DNA, e.g., intact vs. degraded? Third, what fraction of these gene fragments have an abnormal (e.g., mutant) DNA sequence? And fourth, how does this fraction vary with stage of disease? To answer these questions, it is necessary to develop technologies that can simultaneously quantify the number of normal and mutant DNA molecules in a given sample, even when the fraction of mutant molecules is very small. Such sensitive and accurate assays for the detection and quantification of rare variants among a large excess of normal sequences have important applications in many areas of biomedical research. Examples in basic scientific research include the analysis of replication fidelity in various in vitro systems and the determination of mutation rates in cells after treatment with mutagens. Examples in clinical medicine include the identification of mutations in the blood, urine, or stool of cancer patients and the identification of fetal DNA sequences in the plasma of pregnant women.
We previously described an approach, called BEAMing (beads, emulsions, amplification, and magnets), which allows the transformation of a population of DNA fragments into a population of beads each containing thousands of copies of the identical sequence. The bead population generated in this fashion has been shown to accurately represent the initial DNA population. Because 108 beads can be generated in a single test tube and analyzed by standard flow cytometry, this technique has the capacity not only to identify genetic variations present in the original DNA population, but also to quantify precisely their number in comparison to wild-type sequences. In addition to their use for discovering such rare variants, beads generated through the BEAMing process provide excellent templates for nucleotide sequencing, for example, sequencing-by-synthesis. The beads can also be used as templates for both the high-throughput methods recently described for this purpose.
The advantages of having as many copies as possible per bead for both flow cytometric and sequencing applications are clear. We estimate that the number of copies per 1-micron bead produced by BEAMing is 104-105. There is a need in the art for a technique that can increase this number by at least two orders of magnitude.